Liquid chromatography (LC), e.g. high performance liquid chromatography (HPLC), is used routinely in analytical chromatography applications for determination of the quality and quantity of analytes in a variety of samples, both chemical and biological. In these chromatographic techniques, separation of a sample including a mixture of components (also termed analytes) is achieved by conveying the sample in a liquid mobile phase through a stationary phase in a column, thereby causing the sample to separate into its components due to different partitioning between the mobile and stationary phases of each of the components (i.e. the components have different partition coefficients). The stationary phase is most commonly in the form of a bed of particles packed within the column. In order to provide high resolution and throughput of samples, high operating pressures are employed in HPLC of typically up to 600 bar.
In order to deliver ultra-high resolution and/or ultra-fast throughput analysis, which is important for quality of data and cost of analysis, ultra high performance liquid chromatography (UHPLC) has been developed, employing the benefits of low system dispersion and ultra-high system operating pressure (typically greater than 1000 bar). UHPLC columns are a critical element for UHPLC solutions. To fully utilize the capability of a UHPLC instrument to achieve ultra-high resolution, ultra-high throughput analysis, the column is typically packed with stationary phase media including small particles, usually smaller than 2 μm (e.g. 1.5 μm). These particles can be totally porous (i.e. bulk porous), superficially porous, or non-porous and the particles may be surface modified for particular applications. Such demanding operating conditions (high pressure and/or high linear velocity) require that the separation column has excellent bed stability.
Traditionally, HPLC columns are packed in a process using one or more organic solvents (herein “solvent packing”). However, there are some drawbacks associated with this approach noted by the inventors. Firstly, the inventors have found that solvent packing often fails to provide HPLC columns that can be operated under 1300 bar pressure or higher, thereby limiting the usage of such pressures (e.g. 1500 bar) in UHPLC (e.g. in the THERMO SCIENTIFIC® VANQUISH® H UHPLC system). Secondly, most organic solvents are to some extent toxic and/or flammable, thus imposing health and safety hazards on operators and pollution on the environment. Thirdly, the use of organic solvents is expensive due to costs associated with the manufacturing process and the required waste treatment.